JPH0449236A - Phospholipase a2 inhibitor containing nicardipine - Google Patents

Abstract

PURPOSE:To provide a strong phospholipase A2 inhibitor not inactivated by oral administrations and not causing adverse actions such as tunica mucosa damages in oral administration by compounding nicardipine as an active ingredient. CONSTITUTION:The inhibitor contains nicardipine: 2,6-dimethyl-4-(3'-nitrophenyl) -1, 4-dihydropyridine-3, 5-dicarboxylic acid 3-methyl ester-5-beta- (N-benzyl-N- methylamino)ethyl ester of the formula or a salt thereof as an active ingredient. The compound of the formula has excellent inhibiting activity against II type phospholipase A and is employed as an antiinflammatory drug for various inflammations, an antirheumatic drug, an allergic disease therapeutic agent for asthma, atopic diseases, etc., and that for ischemic blood vessel disorders, ulcer, sepsis and pancreatitis which are all related to the phospholipase A2.

Description

Detailed Description of the Invention The present invention relates to 2,6-dimethyl-4-(3'-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-methyl ester -5-β-(N-benzyl-N
The present invention relates to a phospholipase A2 inhibitor containing -methylamino)ethyl ester or a salt thereof as an active ingredient.

Phospholipase A2 inhibitors suppress the production of arachidonic acid and purine lipids from in vivo cell membrane phospholipids by inhibiting the enzymatic activity of phospholipase A2, and inhibit the generation of arachidonic acid and purine lipids from in vivo cell membrane phospholipids, and inhibit the production of these metabolites such as prostaglasins and purine lipids, which are involved in inflammation. This is intended to suppress the production of mediators such as leukotrienes.

Among the phospholipase A2 inhibitors known so far, the one that shows the most potent inhibitory activity is a steroid anti-inflammatory agent (
glucocorticoids). However, it is well known that glucocorticoids exhibit serious side effects.

On the other hand, non-steroidal anti-inflammatory drugs such as indomethacin show anti-inflammatory activity by inhibiting cyclooxygenase of the arachidonic acid cascade and suppressing the production of prostaglacin E2, but they inhibit the gastric mucosa of prostaglacin E2. It is well known that it also impairs the protective effect and is accompanied by side effects such as gastric mucosal disorders.

According to recent research on phospholipase A2 inhibitors, there are two types of phospholipase A2 that differ in their secondary structure and substrate specificity: type I and type 2 phospholipase^2.
The presence of RA was recognized, and phospholipase A2 in the gastric mucosa
It has been shown that phospholipase A2 is the type I phospholipase A2, which is identical to that of the pancreas, and it is thought that the enzyme involved in inflammation is the type II found in other tissues. It's getting stronger.

Therefore, if it shows inhibitory activity against type 2 phospholipase A2, it is considered that it can be used as an anti-inflammatory agent without side effects such as gastric mucosal damage.

Until now, various phospholipase A2 inhibitors have been known, including some that exhibit inhibitory activity against type 2 phospholipase A2. These include problems such as loss of efficacy or weak inhibitory activity.

Therefore, there is a strong need for the development of an orally administrable and potent 2-type phospholipase inhibitor.

-4. It is known that nifedipine and nisoldipine, which are marketed as dihydropyridine calcium antagonists, exhibit inhibitory activity against phospholipase A2 [ln
flammation, Vol. 11. N(1
3353 (1987)).

According to research conducted by the present inventors, as is clear from the experimental examples described below, these drugs have weak inhibitory activity against type 1 phospholipase 82, and it has been confirmed that these drugs cannot overcome the above-mentioned problems. Furthermore, as shown in this report, phospholipase^2 and calcium antagonistic activity are independent, and even compounds that have calcium antagonistic activity have phospholipase Az (including type ■) inhibitory activity. It was also confirmed that this is not necessarily the case.

Based on this state of the art for 1W°, we used type II phospholipase^2 as a screening enzyme and confirmed the inhibitory activity of various compounds against it, and found that 2,6-dimethyl-
4-(3'-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-methyl ester-5
- It has been found that β-(N~hensyl-N-methylamino)ethyl ester or its salt exhibits a significant 2-type inhibitory activity against type 2 phospholipase compared to conventionally known dihydropyridine calcium antagonists such as nifedipine. , which led to the present invention.

2.6-dimethyl-4-(3'-nitrophenyl)-L
4-dihydropyridine-3,5-dicarboxylic acid 3-methyl ester-5-β-(N-benzyl-N-methylamino)ethyl ester is represented by the following structural formula and is called nicardipine, and its hydrochloric acid The salt is called nicardipine hydrochloride.

Nicardipine salts, including nicardipine and nicardipine hydrochloride, were synthesized for the first time by researchers at the applicant's company, and have vasodilatory effects, blood pressure land effects, and antispasmodic effects. It is well known that it is useful as a cerebral vasodilator (Japanese Patent Publication No. 1987-
6417), and it is also known that these effects are based on calcium antagonism. Furthermore, nicardipine hydrochloride has already been widely used clinically as a therapeutic agent for cerebral blood flow disorders such as after-effects of cerebral hemorrhage and after-effects of cerebral infarction, and hypertension.

It is also known that nicardipine and its salts have lipid-lowering and anti-arteriosclerosis effects (Japanese Unexamined Patent Publication No. 59-716
No. 3).

However, it is completely unknown that nicardipine and nicardipine salts, including nicardipine hydrochloride, are useful as inhibitors of phospholipase 82, particularly type 1 phospholipase A2.

An object of the present invention is to provide a phospholipase A2 inhibitor characterized by containing nicardipine or a salt thereof.

The medicament of the present invention will be described in detail below.

The nicardipine of the present invention forms salts. The active ingredients of the pharmaceutical of the present invention include pharmacologically acceptable salts of nicardipine, and specific examples of the salts include sulfuric acid, nitric acid, phosphoric acid, and hydrogen bromide in addition to the above-mentioned hydrochloride. Mineral acids, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, tartaric acid, carbonic acid, salicylic acid, gallic acid, picric acid, methane Examples include acid addition salts and ammonium salts with organic acids such as sulfonic acid and ethanesulfonic acid, and acidic amino acids such as glutamic acid and aspartic acid. Furthermore, nicardipine and its salts have an asymmetric carbon atom and exist as optical isomers. The pharmaceutical compounds of the present invention include isolated optical isomers and mixtures thereof.

Synthesis methods for these compounds are disclosed in various documents such as the above-mentioned Japanese Patent Publication No. 56-6417, and all of these known methods, especially a method applying Hantsch's 1,4-dihydropyridine synthesis method, can be used. It can be easily obtained by applying it.

Next, the phospholipase A2 inhibitory effect of the compounds of the present invention will be explained by giving experimental examples.

Test method for measuring phospholipase A2 inhibitory activity Journal of Biological Chemistry (J
, Biol, Chem, 26 Shiku9), 42
39-4246 (1986)), and was measured by the following method.

22.5 μl of rabbit platelet-derived phospholipase A is added to 100 μl of 135 mM Lis-HCl buffer (pH 8,0) containing 13.5++M calcium chloride and 270 μg/ld bovine serum albumin, and incubated in water for 30 minutes.

Next, 10μ of the compound of the present invention! , and 25μ of E. coli autoflaved specimen labeled with tritium-labeled oleic acid.
Add f (approximately 200,000 cpm) to the reaction solution and react at 6°C for 10 minutes. The reaction is stopped by adding 50 μl of 2N hydrochloric acid. 20■/ll11 after reaction stop! 50 μl of fresh serum albumin was added thereto, and the mixture was left on ice for 30 minutes, centrifuged, and the counts of the centrifuged supernatant were measured.

The autoflaved sample of E. coli labeled with tritium-labeled oleic acid, which was used as a substrate for the measurement of phospholipase A2 inhibitory activity, was prepared as follows. - Add the night-inoculated E. coli culture to 100% tryptone medium (1% doryptone - 0.5% sodium chloride) and incubate at 37°C for 0D5s.

Incubate until 0.4. Next Br
Add 1/100 amount of 1j 35 (surfactant) and tritium-labeled oleic acid 5+++C4, continue incubation at 37°C for 5 hours, and then incubate at 120°C20.
Autoflake at 4°C for 20 minutes at 20°C. Thereafter, the bacterial cells were thoroughly washed with 0.7M Lis hydrochloride buffer containing 0.1% bovine serum albumin and 101M calcium chloride, and then washed with 0.7M Lis hydrochloride buffer containing 0.2% sodium azide and 10mM calcium chloride. Suspend in hydrochloric acid buffer,
Store at 4°C until use. The IC6° values of the phospholipase A2 inhibitory activity of the compounds of the present invention measured by this method are shown in the table.

Measurement of inhibitory effect on mouse ear edema A test sample is applied to both ears of a male ICR mouse (body weight 30 to 35 g), and 30 minutes later, 1 μg/ear of phorbol is applied only to the right ear. After 4 hours, cut out each ear and measure its weight.

The results are shown in the table below together with the PLA2 inhibitory effect.

Correlation between Ca” antagonism and PLA2 inhibition or TPA ear edema suppression ICso (4/M) EDso (μg/ear
) (nifedipine) 〉100 〉300 Nitrendipine 〉100 〉300 Nicardipine 45
230 Nicardipine hydrochloride is also a low toxicity compound, which was confirmed by acute toxicity studies in mice and rats.

The results are shown in the table below.

As is clear from the above experimental results, the pharmaceutical compound of the present invention has excellent inhibitory activity and anti-inflammatory activity, especially against Type 1 phospholipase A2, and does not lose its activity upon oral administration, causing side effects such as gastric mucosal damage. It is useful as a potent phospholipase A2 inhibitor capable of suppressing the biosynthesis of both arachidonic acid and lysophospholipids without the presence of arachidonic acid and lysophospholipid biosynthesis.

Therefore, the phospholipase A2 inhibitor of the present invention is an anti-inflammatory agent for various inflammations, an anti-rheumatic agent, a therapeutic agent for allergic diseases such as asthma and atopic diseases, and a phospholipase A2 inhibitor.
It is used as a therapeutic agent for ischemic vascular disorders, ulcers, sepsis, pancreatitis, etc., which are said to be associated with P. 2.

Administration method, dosage Phosphocontaining nicardipine or its salt 1. The A2 inhibitor above the + bar is particularly effective even when administered orally, so it can be used in oral preparations such as tablets, capsules, powders, fine granules, etc.
It is preferable to make it into granules, gunpowder, oral liquid, etc.
This does not preclude parenteral or sublingual preparations such as injections, suppositories, ointments, emulsions, patches, and nasal preparations, and local administration for skin diseases such as allergic dermatitis. is preferred.

Such formulations can be prepared by conventional formulation methods in the art using commonly used pharmaceutical carriers, excipients, and other additives, but especially those that can be administered once a day or for three days. Publication number 64-7047, patent application No. 2-
The formulation described in No. 66190 is suitable for reducing the burden on patients due to administration and improving compliance.

The dosage of the active ingredient of the pharmaceutical of the present invention is appropriately determined in consideration of the symptoms, weight, age, gender, etc. of the patient to whom it is applied.
Orally 5 to 500 μg per day for adults, preferably 1
The dose is 0 to 250 mg, 1 to 200 mg, preferably 5 to 100 mg, administered intravenously, once or twice a day, as described above, or divided into 3 to 4 doses as usual.

Example 1 Tablets containing 10 μm of nicardipine hydrochloride in one tablet were prepared according to the following formulation.

Nicardipine hydrochloride ・・・・・・・・・
10g lactose・
・・・・・・・・・80g starch
29g Magmagnesium Stearate 1g Tablet making: The above nicardipine hydrochloride is pulverized and mixed with lactose and starch.

The mixture is made into granules by a conventional method, magnesium stearate is added, and 1000 tablets each weighing 0.12 g are compressed.

Example 2 A sterile injectable solution containing nicardipine hydrochloride in 0.1 μm was prepared according to the following formulation.

Nicardipine hydrochloride ・・・・・・・・・
lO ■ Sodium chloride ・・・・・・・
・・900■ Distilled water for injection ・・・・
Preparation of LooInl: Dissolve the above nicardipine hydrochloride in approximately 80 m of distilled water for injection, and then add distilled water for injection to the resulting solution to make a total volume of 100 m. This is then sterile filtered.

Dispense the obtained sterile injection solution into 4 bottles (shielded from light), Seal.

Claims (1)

[Claims] Nicardipine [2,6-dimethyl-4-(3'-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-methyl ester-5-β-(N-benzyl-N-methylamino)ethyl A phospholipase A_2 inhibitor containing ester] or a salt thereof as an active ingredient.